Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

The distance between thiol groups in the γ subunit of coupling factor 1 influences the proton permeability of thylakoid membranes

Spinach chloroplast thylakoids treated in the light with bifunctional maleimides were previously shown to be uncoupled. The increase in proton permeability by these reagents is caused by the cross-linking of an accessible group on the subunit of coupling factor 1 (CF₁) to a group that becomes exposed to reaction with maleimides only when the thylakoids are energized. In this study, several bifunctional maleimides, includingo-,m-, andp-phenylenebismaleimides, 2,3- and 1,5-naphthalenebismaleimides, and azophenylbismaleimide, were tested for their ability to form cross-links and to uncouple photophosphorylation. These reagents form cross-links from about 6 to 19 Å. Each reagent was found to form cross-links in the light and to inhibit photophosphorylation. However, the effectiveness of these compounds as uncouplers decreased as the distance between the cross-linked groups increased, indicating that the distance between two groups on the subunit of CF₁ can regulate proton flux through the membrane. Monofunctional maleimides cause a light-dependent energy transfer type of inhibition of photophosphorylation. Although this inhibition was correlated to the reaction of the maleimide with a group on the subunit that is exposed only in energized thylakoids, the accessible group on this subunit was also modified by the reagent. However, we show here that the accessible group plays no role in the inhibition of photophosphorylation. This group may be blocked by incubating thylakoids in the dark with methyl methanethiolsulfonate. The light-dependent inhibition of photophosphorylation byN-ethylmaleimide was unaffected by this treatment or by the subsequent removal of the methanethiol moiety from the accessible group.     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.     

Supramolecular architecture of cyanobacterial thylakoid membranes: How is the phycobilisome connected with the photosystems?

Cyanobacteria, as the most simple organisms to perform oxygenic photosynthesis differ from higher plants especially with respect to the thylakoid membrane structure and the antenna system used to capture light energy. Cyanobacterial antenna systems, the phycobilisomes (PBS), have been shown to be associated with Photosystem 2 (PS 2) at the cytoplasmic side, forming a PS 2-PBS-supercomplex, the structure of which is not well understood. Based on structural data of PBS and PS 2, a model for such a supercomplex is presented. Its key features are the PS 2 dimer as prerequisite for formation of the supercomplex and the antiparallel orientation of PBS-cores and the two PS 2 monomers which form the contact area within the supercomplex. Possible consequences for the formation of superstructures (PS 2-PBS rows) within the thylakoid membrane under so-called state 1 conditions are discussed. As there are also indications for specific functional connections of PBS with Photosystem 1 (PS 1) under so-called state 2 conditions, we show a model which reconciles the need for a structural interaction between PBS and PS 1 with the difference in structural symmetry (2-fold rotational symmetry of PBS-cores, 3-fold rotational symmetry of trimeric PS 1). Finally, the process of dynamic coupling and uncoupling of PBS to PS 1 and PS 2, based on the presented models, shows analogies to mechanisms for the regulation of photosynthetic electron flow in higher plants-despite the very different organization of their thylakoid membranes in comparison to cyanobacteria.Abbreviations APC allophycocyanin - b ₆ f cytochrome b ₆ f complex - CP chlorophyll protein - FNR ferredoxin-NADP⁺-oxidoreductase - LD linkerprotein-domain - LHC light-harvesting complex - Pc plastocyanin - PC phycocyanin - PD phycobiliprotein-domain - PS 1 Photosystem 1 - PS 2 Photosystem 2 - PBS phycobilisome Dedicated to Prof. Dr. Horst Senger on the occasion of his 65th birthday.     

Functional characterization of the PS II–LHC II supercomplex isolated by a direct method from spinach thylakoid membranes

Recently, a novel procedure to isolate a highly pure and active Photosystem II preparation directly from thylakoid membranes, referred to as PS II–LHC II supercomplex, was reported [Eshaghi et al. (1999) FEBS Lett 446: 23–26]. In addition to the reaction center core proteins, the supercomplex contains all the extrinsic proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. In this paper, the functional properties of this isolated supercomplex are further characterized by using EPR spectroscopy, thermoluminescence, fluorescence relaxation kinetics and flash induced oxygen yield measurements. The PS II–LHC II supercomplex contains, in addition to Q_A and Q_B, a small pool of plastoquinone (PQ). Although the isolated complex is no longer membrane bound, it has preserved functional characteristics of a well defined PS II preparation with the exception of some modification of Q_B sites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Does the speed of shortening affect steady-state force depression in cat soleus muscle?

It has been stated repeatedly for the past 50 years that the steady-state force depression following shortening of an activated muscle depends on the speed of shortening. However, these statements were based on results from experiments in which muscles were shortened at different speeds but identical activation levels. Therefore, the force during shortening was changed in accordance with the force-velocity relationship of muscles: that is, increasing speeds of shortening were associated with decreasing forces, and vice versa. Consequently, it is not possible at present to distinguish whether force depression is caused by the changes in speed, as frequently stated, or the associated changes in force, or both. The purpose of this study was to test if force depression depends on the speed of shortening. We hypothesized that force depression was dependent on the force but not the speed of contraction. Our prediction is that the amount of force depression after shortening contractions at different speeds could be similar if the force during contraction was controlled at a similar level. Cat soleus muscles (n=7) were shortened by 9 or 12 mm at speeds of 3, 9, and 27 mm/s, first with a constant activation during shortening (30Hz), then with activation levels that were reduced (<30Hz) for the slow speeds (3 and 9 mm/s) to approximate the shortening forces of the fast speed contractions (27 mm/s). If done properly, force depression could be precisely matched at the three different speeds, indicating that force depression was related to the force during the shortening contraction but not to the speed. However, in order to match force depression, the forces during shortening had to be systematically greater for the slow compared to the fast speeds of shortening, suggesting that force depression also depends on the level of activation, as force depression at constant activation levels can only be matched if the force during shortening, evaluated by the mechanical work, is identical. Therefore, we conclude that force depression depends on the force and activation level during shortening, but does not depend on the speed of shortening as has been assumed for half a century. These results support, but do not prove, the current hypothesis that force depression is caused by a stress-related cross-bridge inhibition in the actin-myosin overlap zone that is newly formed during muscle shortening.     

Comparison of the α and β isomeric forms of the detergent n-dodecyl-D-maltoside for solubilizing photosynthetic complexes from pea thylakoid membranes

Mild non-ionic detergents are indispensable in the isolation of intact integral membrane proteins and protein-complexes from biological membranes. Dodecylmaltoside (DM) belongs to this class of detergents being a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric center of the carbohydrate head group, axial in α-DM and equatorial in β-DM. In this paper, we have investigated the solubilizing properties of α-DM and β-DM on the isolation of photosynthetic complexes from pea thylakoids membranes maintaining their native architecture of stacked grana and stroma lamellae. Exposure of these stacked thylakoids to a single step treatment with increasing concentrations (5-100mM) of α-DM or β-DM resulted in a quick partial or complete solubilization of the membranes. Regardless of the isomeric form used: 1) at the lowest DM concentrations only a partial solubilization of thylakoids was achieved, giving rise to the release of mainly small protein complexes mixed with membrane fragments enriched in PSI from stroma lamellae; 2) at concentrations above 30mM a complete solubilization occurred with the further release of high molecular weight protein complexes identified as dimeric PSII, PSI-LHCI and PSII-LHCII supercomplexes. However, at concentrations of detergent which fully solubilized the thylakoids, the α and β isomeric forms of DM exerted a somewhat different solubilizing effect on the membranes: higher abundance of larger sized PSII-LHCII supercomplexes retaining a higher proportion of LHCII and lower amounts of PSI-LHCI intermediates were observed in α-DM treated membranes, reflecting the mildness of α-DM compared with its isomer. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Bioinformatic search for plant homologs of the protein kinase Bub1—a key component of the mitotic spindle assembly checkpoint

The bioinformatic search identified 14 plant homologs of the mitotic spindle assembly checkpoint protein kinase Bub1 of animals, yeasts, and myxomycetes. It was demonstrated that the closest plant homologs of the protein kinase Bub1 were proteins with previously unknown features: XP_002274770.1 (CBI21878.1) from Vitis vinifera, EEC82122.1 from Oryza sativa Indica, EEE67244.1 from O. sativa Japonica, EEF44403.1 from Ricinus communis, and CAL57156.1 from Ostreococcus tauri. The simulation and analysis of spatial structures of the catalytic domains of the EEC82122.1, EEE67244.1, and XP_002274770.1 (CBI21878.1) proteins confirmed their conformity with the spindle assembly checkpoint protein kinases Bub1.     

Storage of light-driven transthylakoid proton motive force as an electric field (Δψ) under steady-state conditions in intact cells of <Emphasis Type="Italic">Chlamydomonasreinhardtii</Emphasis>

Proton motive force (pmf) is physiologically stored as either a ΔpH or a membrane potential (Δψ) across bacterial and mitochondrial energetic membranes. In the case of chloroplasts, previous work (Cruz et al. 2001, Biochemistry 40: 1226–1237) indicates that Δψ is a significant fraction of pmf, in vivo, and in vitro as long as the activities of counterions are relatively low. Kinetic analysis of light-induced changes in the electrochromic shift (ECS) in intact leaves was consistent with these observations. In this work, we took advantage of the spectroscopic properties of the green alga, Chlamydomonas reinhardtii, to demonstrate that light-driven Δψ was stored in vivo over the hours time scale. Analysis of the light-induced ECS kinetics suggested that the steady-state Δψ in 400 μmol photons m⁻² s⁻¹ red light was between 20 and 90 mV and that this represented about 60% of the light-induced increase in pmf. By extrapolation, it was surmised that about half of total (basal and light-induced) pmf is held as Δψ. It is hypothesized that Δψ is stabilized either by maintaining low chloroplast ionic strength or by active membrane ion transporters. In addition to the strong implications for regulation of photosynthesis by the xanthophyll cycle, these results imply that pmf partitioning is important across a wide range of species.     

The asymmetry of existence: do we owe our existence to cold dark matter and the weak force?

A common theme throughout biology is homochirality, including its origin and especially implications. Homochirality has also intrigued scientists because of the hypothesis that life, as it currently exists, could not have occurred without it. In this review, we discuss several hypotheses regarding homochirality and their linkage to processes that range from subatomic in scale to processes that help define the structure of the universe. More importantly, this exploration begins with the knowledge that humans inhabit the universe in which there is an excess of normal matter over antimatter. It is a universe characterized by homochirality but is nonetheless contained in what is most easily described as a 3+1 dimensional spacetime wherein most laws of physics are invariant under spacetime transformations. This restriction on spacetime poses significant constraints on the processes that can be invoked to explain homochirality. However, in dealing with such restraints, including the total mass contained in the universe, the concepts of cold dark matter and dark energy can be incorporated into cosmological models with resultant behaviors and predictions very much in accord with the findings of the cosmic background surveys. Indeed, the introduction of cold dark matter and dark energy to solve problems relating to the mass found in the universe may provide a means for generating the needed asymmetry to allow homochirality to arise.     

Chymotrypsinogen · inositol phosphatide complexes and the transport of digestive enzyme across membranes

Chymotrypsinogen A was almost quantitatively extracted from aqueous solution in the presence of inositol phosphatides at relatively low concentrations of both ligands. Calcium ion facilitated the interaction at concentrations of 10^?4–10^?5 M. A water-insoluble chymotrypsinogen · Ca²⁺ · inositol phosphatide complex was formed with an apparent stochiometry of 3 mol phospholipid : 3 mol Ca²⁺ : 1 mol protein. Small changes in the structure of the protein prevented complex formation; in particular, the almost identical α-chymotrypsin, did not form complexes under the conditions studied. On the other hand, an homologous, but structurally substantially different, secretory protein, trypsinogen, did form complexes. Water-insoluble complexes were not formed with albumin, carbonic anhydrase or lactic dehydrogenase under the same circumstances. Neither phosphatidylethanolamine nor phosphatidylcholine formed complexes with chymotrypsinogen. Phosphatidylserine formed complexes, but was less effective than inositol phosphatides. Complex formation and stability was dependent upon “critical” concentrations of both Ca²⁺ and H⁺. Extraction of the protein from solution increased from neglible to complete when the calcium concentration of the medium was raised slightly from 1.0 · 10^?4 M to 1.5·10^?4 M. Conversely, dissociation was complete when H⁺ concentration was decreased slightly from pH 6.5 to 7.0. The complex is apparently formed as the result of specific electrostatic interactions between the polar head group of the inositol phosphatide and the protein, with the nonpolar alipathic fatty acid chains of the phospholipid providing a hydrophobic microenvironment for the protein. It is proposed that such complexes could account for the movement of digestive enzyme through membranes.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

High-affinity binding of fungal β-glucan elicitors to cell membranes of species of the plant family Fabaceae

Microsomal preparations of six species of the plant family Fabaceae were screened for high-affinity binding of branched (1 3), (1 6)--glucans. Oligoglucosides of this type are specific elicitors of phytoalexin accumulation in soybean (Glycine max L.), a member of this family. The species studied were alfalfa (Medicago sativa L.), broadbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A ¹²⁵I-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 3), (1 6)--glucan fraction with an average degree of polymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural complexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 3), (1 6) hepta--glucoside for which soybean binding sites display highest affinity. Radioligand competition experiments against unlabeled fungal -glucan resulted in the identification of high-affinity binding in alfalfa, bean, lupin, and pea. Half-maximal competition concentrations (IC₅₀) for fungal -glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled hepta--glucoside showed that for alfalfa, lupin and pea the IC₅₀ values for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC₅₀ significantly higher than soybean (68 nM) suggesting that the structural motif recognized by its binding sites is not identical to that of soybean or the other three species. Radioligand saturation assays with alfalfa, lupin and pea microsomes using an ₁₂₅I-labeled aminophenylethylamine hepta--glucoside conjugate gave dissociation constants (K_d) of 5.3, 3.7, and 1.8 nM, respectively. The affinity of these sites for hepta- glucoside was in the same range as that of soybean (K_d 1–3 nM), whereas the affinity of the binding sites of bean for the same ligand was significantly lower (K_d = 33 nM). Good correlation was found between the presence of high-affinity binding and the accumulation of isoflavonoid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal -glucan. Lupin displayed a strong wound-induced accumulation of prenylated isoflavones which was independent of the presence of -glucan, making it impossible to determine phytoalexin induction in response to elicitor. No specific binding or phytoalexin accumulation in response to glucans was observed in broadbean. This is the first report on the existence of possibly homologous elicitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathogen perception mechanisms in plants.Abbreviations DP degree of polymerization - EC₅₀ concentration of elicitor necessary to obtain a half-maximal biological response - HG synthetic (1 3), (1 6)-hepta--glucoside phytoalexin elicitor - HG-APEA 1-[4-(2-aminophenyl)ethylamino-1-hexaglucosyl]deoxyglucitol - IC₅₀ ligand concentration necessary to obtain half-maximal displacement of radioligand in competition binding assays - K_d dissociation constant - OS branched (1 3), (1 6)--glucan obtained by hydrolysis of mycelial walls of Phytophthora sojae - OS-APEA 1-[4-(2-armnophenyl)ethylamino-1-oligoglucosyl]deoxyglucitol conjugate of OS This work was supported by the Comision Interministerial de Ciencia y Tecnologia grant BI091-0366 (E.G.C.), the Volkswagen-Stiftung (E.G.C. and J.E.), the Deutsche Forschungsgemeinschaft, SFB-369 (J.E.), the Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (J.E.), Fonds der Chemischen Industrie (J.E.) and the EU Human Capital and Mobility Program (J.E. and E.G.C.).     

Plant–soil feedbacks of exotic plant species across life forms: a meta-analysis

Invasive exotic plant species effects on soil biota and processes in their new range can promote or counteract invasions via changed plant–soil feedback interactions to themselves or to native plant species. Recent meta-analyses reveale that soil influenced by native and exotic plant species is affecting growth and performance of natives more strongly than exotics. However, the question is how uniform these responses are across contrasting life forms. Here, we test the hypothesis that life form matters for effects on soil and plant–soil feedback. In a meta-analysis we show that exotics enhanced C cycling, numbers of meso-invertebrates and nematodes, while having variable effects on other soil biota and processes. Plant effects on soil biota and processes were not dependent on life form, but patterns in feedback effects of natives and exotics were dependent on life form. Native grasses and forbs caused changes in soil that subsequently negatively affected their biomass, whereas native trees caused changes in soil that subsequently positively affected their biomass. Most exotics had neutral feedback effects, although exotic forbs had positive feedback effects. Effects of exotics on natives differed among plant life forms. Native trees were inhibited in soils conditioned by exotics, whereas native grasses were positively influenced in soil conditioned by exotics. We conclude that plant life form matters when comparing plant–soil feedback effects both within and between natives and exotics. We propose that impact analyses of exotic plant species on the performance of native plant species can be improved by comparing responses within plant life form.